The present invention provides novel antibody compositions and negative selection processes for enriching human cells from murine/human chimeric haematopoietic cell suspension.
Human cells are often transplanted into mice in order to study various diseases as the study of human cells in such diseases cannot always be adequately modelled in vitro.
Human/Murine transplant chimeras have been used to study human autoimmune diseases, such as Graves disease (Yoshikawa, N. et al., 1997; Weetman, A. P., 1996). Chimeric mice have also been used to evaluate the efficacy of anti-viral agents in the treatment of human immunodeficiency virus (HIV) and Epstein Barr Virus (EBV) (Jenkins M. et al., 1998; Fuzzati-Armentero, M. T., 1998). However, in such models it has been difficult to efficiently retrieve human cells from the chimeric mice to enable further assays.
Human/Murine chimeric mice are also used to study hematopoietic stem cells. The hematopoietic stem cell is identified by its distinct functional capabilities, including self-renewal and long-term repopulation of all hematopoietic lineages. In vitro assays, such as long-term culture-initiating cells (LTC-IC) (Sutherland, H. J., et al., 1989) are not entirely predictive of repopulating and homing potential in vivo and therefore, several groups have transplanted human hematopoietic stem cells into RAGxe2x88x92/xe2x88x92 (Koyanagi, Y. et al., 1997) or severe combined immune deficiency (SCID) mice (McCune, J. M. et al., 1988; Kamel-Reid S. and Dick J. E., 1988; Kyoizumi S. et al., 1992; Larochelle, A., et al., 1996); or non obese diabetic SCID (NOD.SCID) (Cashman, J. D., et al. 1997). The surviving transplanted human cells and their progeny may be very rare in bone marrow and blood. This creates difficulties in determining the success of engraftment and makes further functional assay of the surviving engrafted human cells not feasible. Therefore, there is a need to develop a method to enrich for human cells allowing the detection and isolation of these low frequency human cells in chimeric SCID/Hu, NOD.SCID/Hu or RAGxe2x88x92/xe2x88x92/Hu mice.
The present inventors have developed an antibody composition for use in enriching human cells from human-murine chimeric hematopoietic cell suspensions. The antibodies in the antibody composition are specific for selective markers associated with murine cells.
In particular, the present inventors have found that a negative selection technique using an antibody composition containing an antibody specific for murine leukocytes (such as anti-CD45 and/or anti major histocompatibility complex class I (MHC-I)) alone or in combination with an antibody capable of binding to murine erythroid cells gives a cell preparation highly enriched for human cells. Accordingly, the present invention provides an antibody composition comprising an antibody specific for murine leukocytes and an antibody specific for murine erythroid cells.
Preferably, the present invention provides an antibody composition comprising an antibody specific for murine CD45 and an antibody capable of binding to a murine erythroid cells. CD45 is a pan-leukocyte maker expressed on all hematopoietic cells with the exception of erythroid cells (Ledabetter, J.A and Herzenburg, L A., Immunol. Rev. 47:63 (1979); Thomas, M. L. Ann. Rev. Immunol. 7:339-369 (1989); Van Ewijk, W. et al., J. Immnumol. 127:2594 (1981)). Human cells will be enriched from human-murine chimeric hematopoietic cell suspension by depleting only CD45+ murine cells although the presence of murine erythroid cells will limit the degree of enrichment. Combining an antibody which recognizes murine erythroid cells with an anti-murine CD45 antibody will deplete all murine hematopoietic cells and thereby achieve extensive enrichment of human cells thus enabling detection and further study.
The enrichment and recovery of human cells using the antibody composition of the invention in a negative selection technique has many advantages over conventional positive selection techniques. Most importantly, the recovered human cells are not labelled or coated with antibodies thereby making them highly suitable for further study.
The present invention includes a negative selection process for enriching and recovering human cells in a sample containing human cells and murine cells comprising:
(a) reacting the sample with an antibody composition containing antibodies capable of binding to murine leukocytes under conditions such that conjugates are formed between the antibodies and murine leukocytes;
(b) removing the conjugates; and
(c) recovering a cell population which is enriched in human cells and depleted of murine leukocytes.
In a preferred embodiment, the present invention provides
(a) reacting the sample with an antibody composition containing antibodies capable of binding to murine leukocytes and antibodies capable of binding to murine erythroid cells under conditions such that conjugates are formed between the antibodies and the murine leukocytes and murine erythroid cells;
(b) removing the conjugates; and
(c) recovering a cell population which is enriched in human cells and depleted of murine leukocytes and murine erythroid cells.
The present invention also includes a kit useful in performing the process of the invention comprising antibodies specific for murine leukocytes and murine erythroid cells and instructions for performing the process of the invention.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.